Expression of Eukaryotic Cytochromes P450 in E. co/i

Jenkins, Christopher M.; Pikuleva, Irina A.; Waterman, Michael R.

doi:10.1385/0-89603-519-0:181

Cited by 20 publications

(27 citation statements)

References 10 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…E. coli NADPH:flavodoxin reductase (encoded by fpr) and flavodoxin (encoded by fld) were cloned and overexpressed in E. coli, and cell lysates were added to in vitro reactions with terpene synthases and P450s. The E. coli reductase system has been shown previously to support P450 activity in vitro (24,25), but it did not facilitate sesquiterpene oxidation in assays with the two Nostoc P450s. Under the assumption that an NADPH: ferredoxin reductase and ferredoxin pair from Nostoc may reconstitute a native reductase system for P450NS and P450NP, both proteins were cloned from Nostoc sp.…”

Section: Resultsmentioning

confidence: 84%

Identification of Sesquiterpene Synthases fromNostoc punctiformePCC 73102 andNostocsp. Strain PCC 7120

et al. 2008

View full text Add to dashboard Cite

Cyanobacteria are a rich source of natural products and are known to produce terpenoids. These bacteria are the major source of the musty-smelling terpenes geosmin and 2-methylisoborneol, which are found in many natural water supplies; however, no terpene synthases have been characterized from these organisms to date. Here, we describe the characterization of three sesquiterpene synthases identified in Nostoc sp. strain PCC 7120 (terpene synthase NS1) and Nostoc punctiforme PCC 73102 (terpene synthases NP1 and NP2). The second terpene synthase in N. punctiforme (NP2) is homologous to fusion-type sesquiterpene synthases from Streptomyces spp. shown to produce geosmin via an intermediate germacradienol. The enzymes were functionally expressed in Escherichia coli, and their terpene products were structurally identified as germacrene A (from NS1), the eudesmadiene 8a-epi-␣-selinene (from NP1), and germacradienol (from NP2). The product of NP1, 8a-epi-␣-selinene, so far has been isolated only from termites, in which it functions as a defense compound. Terpene synthases NP1 and NS1 are part of an apparent minicluster that includes a P450 and a putative hybrid two-component protein located downstream of the terpene synthases. Coexpression of P450 genes with their adjacent located terpene synthase genes in E. coli demonstrates that the P450 from Nostoc sp. can be functionally expressed in E. coli when coexpressed with a ferredoxin gene and a ferredoxin reductase gene from Nostoc and that the enzyme oxygenates the NS1 terpene product germacrene A. This represents to the best of our knowledge the first example of functional expression of a cyanobacterial P450 in E. coli.Terpenoids constitute the largest group of natural products, constituting over 20,000 described compounds (14). These compounds have a wide range of biological functions and are synthesized by plants and microbes as, for example, pigments, hormones and signaling molecules, antibiotics, antifeedants, or pollinator attractants. Many of these compounds are present in minute quantities and have been an important target for synthetic chemists (22, 52) and metabolic engineers (10,35,50).Terpenes are synthesized from linear isoprene diphosphate precursors with various chain lengths, and their enormous structural diversity is the result of the large number of possible enzyme-catalyzed cyclizations and rearrangements that the double-bond-containing isoprene chains can undergo (14, 49). Cyclizations of isoprene diphosphate chains are catalyzed by terpene synthases (also known as terpene cyclases). Depending on the chain length of their isoprene diphosphate substrates, terpene synthases can be classified as mono-, sesqui-, or diterpene synthases, which catalyze the cyclization of geranyl diphosphate (C 10 ), farnesyl diphosphate (FPP; C 15 ), or geranylgeranyl diphosphate, respectively. Well-known examples of cyclic terpenes are the monoterpene 2-methylisoborneol and sesquiterpene geosmin (responsible for the earthy odor of soil and lake water), the trichothecenes (m...

show abstract

Section: Resultsmentioning

confidence: 84%

Identification of Sesquiterpene Synthases fromNostoc punctiformePCC 73102 andNostocsp. Strain PCC 7120

et al. 2008

View full text Add to dashboard Cite

show abstract

“…Heterologous expression of t CYP3A37 in E. coli [Jenkins et al, 1998] was carried out by inoculating 5 ml of Luria-Bertani ampicillin medium with a single colony of DH5 ␣ FIQ E. coli transformants, which was then incubated for 23 h with shaking (150 rpm) at 30 ° C. Levels of P450 expression were determined by reduced CO/reduced difference spectrum (Genesys 6 UV-vis spectrophotometer, Thermo Spectronic, Rochester, NY). E. coli membranes and cytosol were then prepared for AFB 1 metabolism experiments [Yip and Coulombe, 2006].…”

Section: Analysis Of Biological Activitymentioning

confidence: 99%

Structure, Genetic Mapping, and Function of the Cytochrome P450 3A37 Gene in the Turkey <i>(Meleagris gallopavo)</i>

Rawal

Mendoza

Reed

et al. 2009

Cytogenet Genome Res

View full text Add to dashboard Cite

Cytochromes P450 (P450 for protein; CYP for gene) are a superfamily of membrane-bound hemoproteins that oxidize a large number of endogenous and exogenous compounds. Through oxidation reactions, these enzymes are often responsible for the toxic and carcinogenic effects of natural food-borne toxicants, such as the mycotoxin aflatoxin B₁ (AFB₁). Previous studies in our laboratory have shown that the extreme sensitivity of turkeys to AFB₁ is in part explained by efficient hepatic P450-mediated epoxidation to the toxic and reactive metabolite the exo-AFB₁-8,9-epoxide (AFBO). Using 3′-5′-rapid amplification of cDNA ends (RACE), we amplified CYP3A37 from turkey liver RNA, the E. coli-expressed protein which efficiently epoxidates AFB₁. Turkey CYP3A37 has an ORF of 1512 bp, and the protein is predicted to be 504 amino acids with 97% homology to chicken CYP3A37. The turkey gene is organized into 13 exons and 12 introns. A single nucleotide polymorphism in the 11th intron was used to assign CYP3A37 to turkey linkage group 10 (corresponding to chicken chromosome 14, GGA14). Because of the important role of P450s in the extreme sensitivity of turkeys to the toxic effects of AFB₁, this study will contribute to the identifying allelic variants of this important gene in poultry.

show abstract

“…Expression of P450 in E. coli cultures is dependent on numerous experimental factors, not all of which are well understood, including choice of vector, strain background, culture medium, culture time and temperature, medium-to-flask volume ratio [Jenkins et al, 1998], shaker speed [Jansson et al, 2000], antibiotics [Kusano et al, 1999], and so forth. It has also been suggested that high expression of P450 and P450 reductase may reduce bacterial cell viability [Kranendonk et al, 1999;Jansson et al, 2000].…”

Section: P450 Expressionmentioning

confidence: 99%

Recombinant human P450 forms 1A1, 1A2, and 1B1 catalyze the bioactivation of heterocyclic amine mutagens in Escherichia coli lacZ strains

Josephy

Batty

Boverhof

2001

Environ and Mol Mutagen

View full text Add to dashboard Cite

Three recombinant human P450 enzymes, forms 1A1, 1A2, and 1B1, were coexpressed with NADPH-cytochrome P450 reductase in an E. coli lacZ strain suitable for detection of the mutagenicity of heterocyclic and aromatic amines. The resulting strains expressed the recombinant P450 holoenzymes at high levels. MeIQ (2-amino-3,4-dimethylimidazo[4,5-f]quinoline) was activated effectively by P450 1A2, weakly by P450 1A1, and not detectably by P450 1B1. MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline) and Trp-P-2 (3-amino-1-methyl-5H-pyrido[4,3-b]indole) were activated by all three enzymes, with form 1A2 the most effective. These strains facilitate analysis of the substrate specificity of human P450 forms that participate in the metabolic activation of carcinogens.

show abstract

Expression of Eukaryotic Cytochromes P450 in E. co/i

Cited by 20 publications

References 10 publications

Identification of Sesquiterpene Synthases fromNostoc punctiformePCC 73102 andNostocsp. Strain PCC 7120

Identification of Sesquiterpene Synthases fromNostoc punctiformePCC 73102 andNostocsp. Strain PCC 7120

Structure, Genetic Mapping, and Function of the Cytochrome P450 3A37 Gene in the Turkey <i>(Meleagris gallopavo)</i>

Recombinant human P450 forms 1A1, 1A2, and 1B1 catalyze the bioactivation of heterocyclic amine mutagens in Escherichia coli lacZ strains

Contact Info

Product

Resources

About